transplantologiyaТрансплантологияTransplantologiya. The Russian Journal of Transplantation2074-05062542-0909IPO Association of Transplantologiststransplantologiya-97Research ArticleАКТУАЛЬНЫЕ ВОПРОСЫ ТРАНСПЛАНТОЛОГИИACTUAL ISSUES OF TRANSPLANTATIONИспользование магнитных частиц для фиксации изолированных клеток при субретинальной трансплантацииUsing of magnetic particles for fi xing of isolated cells in subretinal transplantationТемновА. А.TemnovA. A.aa-temnov@yandex.ruБелыйЮ. А.BelyyYu. A.МиргородскаяС. А.MirgorodskayaS. A.СеменовА. Д.SemenovA. D.ШацкихА. В.ShatskikhA. V.РевищинА. В.RevishchinA. V.ПавловаГ. В.PavlovaG. V.КустН. Н.KustN. N.СклифасА. Н.SklifasA. N.ГБУЗ «НИИ скорой помощи им. Н.В. Склифосовского ДЗМ», МоскваРоссияN.V. Sklifosovsky Research Institute for Emergency Medicine of Moscow Healthcare Department, MoscowRussian FederationКалужский филиал ФГБУ «МНТК «Микрохирургия глаза» им. акад. С.Н. Федорова», КалугаРоссия«The Interbranch Scientific-Technical Complex «Eye microsurgery» named after academician Fedorov» of the Ministry of Health of Russia, KalugaRussian FederationФГБУ «МНТК «Микрохирургия глаза» им. акад. С.Н. Федорова», МоскваРоссия«The Interbranch Scientific-Technical Complex «Eye microsurgery» named after academician Fedorov» of the Ministry of Health of Russia, MoscowRussian FederationФГБУН Институт биологии гена Российской академии наук, МоскваРоссияInstitute of gene biology of Russian Academy of Sciences, MoscowRussian FederationФГБУН Институт биофизики клетки Российской академии наук, ПущиноРоссияInstitute of сell biophysics Russian Academy of Sciences, PushchinoRussian Federation201405022016041220Copyright © Темнов А.А., Белый Ю.А., Миргородская С.А., Семенов А.Д., Шацких А.В., Ревищин А.В., Павлова Г.В., Куст Н.Н., Склифас А.Н., 20162016Темнов А.А., Белый Ю.А., Миргородская С.А., Семенов А.Д., Шацких А.В., Ревищин А.В., Павлова Г.В., Куст Н.Н., Склифас А.Н.Temnov A.A., Belyy Y.A., Mirgorodskaya S.A., Semenov A.D., Shatskikh A.V., Revishchin A.V., Pavlova G.V., Kust N.N., Sklifas A.N.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.jtransplantologiya.ru/jour/article/view/97Цель

Цель: данное исследование посвящено разработке метода введения магнитных микрочастиц в цитоплазму клеток с их последующей фиксацией под сетчаткой глаза посредством магнитного имплантата.

Материал и методы

Материал и методы. Магнитные частицы (d = 2,8 мкм), обработанные плюрониками, вводили в цитоплазму клеток линии НЕК-293, экспрессирующих GFP. Под общей анестезией с помощью устройства для локального субретинального дозированного введения клетки НЕК-293, содержащие магнитные частицы, вводили субретинально в глаз кролика (96 глаз, 48 кроликов). В опытной группе (48 глаз, 24 кролика) для удержания трансплантированных клеток эписклерально фиксировали магнитный имплантат. В группе контроля (48 глаз, 24 кролика) магнитный имплантат не фиксировали. После проведения хирургических вмешательств в заданные сроки (1-е, 3-и, 7-е, 14-е, 21-е и 30-е сутки) всем животным производили биомикроскопию и офтальмоскопию с фоторегистрацией, ультразвуковое исследование, компьютерную томографию и последующее морфологическое исследование.

Результаты

Результаты. Введение магнитных частиц в цитоплазму клеток линии НЕК-293 не оказывает влияния на жизнеспособность клеток. Клетки НЕК-293, содержащие магнитные частицы, остаются в зоне введения под действием магнитного поля эписклерально фиксированного имплантата в течение 21 суток. Такие же клетки, введенные без последующей фиксации магнитного имплантата, находятся в зоне введения не более 3 суток.

Выводы

Выводы: использование клеток, содержащих магнитные частицы, при фиксации магнитного имплантата может быть перспективным методом клеточной терапии для лечения заболеваний сетчатки.

 

 

Purpose

Purpose: This study focuses on the development of the method of introduction of magnetic microparticles in the cytoplasm of HEK-293 cell line with their subsequent fixation under the retina of the eye.

Materials and Methods

Materials and Methods. Magnetic particles (d = 2,8 mm) were treated with pluronic and injected into the cytoplasm of HEK-293 cell line, expressing GFP. The surgery was made under general anesthesia. HEK-293 containing magnetic particles were injected into the subretinal space of rabbit eyes (eyes 96, 48 rabbits) using original dosing device. In the experimental group (48 eyes, 24 rabbits) we fixed episcleral magnetic implant to hold cells in local place. In the control group (48 eyes, 24 rabbit) magnetic implant was not fixed. After the surgery all animals were examined using biomicroscopy, ophthalmoscopy with photographic recording, ultrasound, computed tomography and morphological study in certain terms (1, 3, 5, 7, 14, 21 day and 1 month).

Results

Results: The introduction of the magnetic particles into the cytoplasm of HEK 293 cell line has no effect on cell viability. HEK-293 containing magnetic particles remains in the place of injection during 21 days in rabbit eyes, where the magnetic implants were fixed (in control group during 3 days).

Conclusions

Conclusions: Using of cells containing magnetic particles with fixation of the magnetic implant can be a promising method for cell therapy for the treatment of retinal diseases.

 

 

НЕК-293магнитные частицыплюроникиHEK-293magnetic particlespluronicsReferencesChaum, E. Retinal neuroprotection by growth factors: a mechanistic perspective / E. Chaum // J. Cell Biochem. – 2003 – Vol. 88, N.1. – P. 57–75.Chaum, E. Retinal neuroprotection by growth factors: a mechanistic perspective / E. Chaum // J. Cell Biochem. – 2003 – Vol. 88, N.1. – P. 57–75.Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging effects of constant light / M.M. LaVail, K. Unoki, D. Yasumura [et al.] // Proc. Natl. Acad. Sci. – 1992 – Vol. 89, N.23. – P. 11249–11253.Multiple growth factors, cytokines, and neurotrophins rescue photoreceptors from the damaging effects of constant light / M.M. LaVail, K. Unoki, D. Yasumura [et al.] // Proc. Natl. Acad. Sci. – 1992 – Vol. 89, N.23. – P. 11249–11253.Protection of mouse photoreceptors by survival factors in retinal degenerations / M.M. LaVail, D. Yasumura, M.T. Matthes[et al.] // Invest. Ophthalmol. Vis. Sci. – 1998. – Vol. 39, N.3. – P. 592–602.Protection of mouse photoreceptors by survival factors in retinal degenerations / M.M. LaVail, D. Yasumura, M.T. Matthes[et al.] // Invest. Ophthalmol. Vis. Sci. – 1998. – Vol. 39, N.3. – P. 592–602.Molecular mechanisms of lightinduced photoreceptor apoptosis and neuroprotection for retinal degeneration / A. Wenzel, C. Grimm, M. Samardzija [et al.] // Prog. Ret. Eye Res. – 2005. – Vol. 24, N.2. – P. 275–306.Molecular mechanisms of lightinduced photoreceptor apoptosis and neuroprotection for retinal degeneration / A. Wenzel, C. Grimm, M. Samardzija [et al.] // Prog. Ret. Eye Res. – 2005. – Vol. 24, N.2. – P. 275–306.Bennett, J. Gene therapy for retinitis pigmentosa / J. Bennett // Curr. Opin.Mol. Ther. – 2000. – Vol. 2, N.4. – P. 420–425.Bennett, J. Gene therapy for retinitis pigmentosa / J. Bennett // Curr. Opin.Mol. Ther. – 2000. – Vol. 2, N.4. – P. 420–425.Borras, T. Recent developments in ocular gene therapy / T. Borras // Exp. Eye Res. – 2003. – Vol. 76, N.6. – P. 643–652.Borras, T. Recent developments in ocular gene therapy / T. Borras // Exp. Eye Res. – 2003. – Vol. 76, N.6. – P. 643–652.Hauswirth, W. Retinal Gene Therapy 1998: Summary of a Workshop / W. Hauswirth, R. McInnes // Mol. Vis. – 1998. – Vol.4. – P.11.Hauswirth, W. Retinal Gene Therapy 1998: Summary of a Workshop / W. Hauswirth, R. McInnes // Mol. Vis. – 1998. – Vol.4. – P.11.Hermens, W.T. Viral vectors, tools for gene transfer in the nervous system / W.T. Hermens, J. Verhaagen // Prog. Neurobiol. – 1998. – Vol. 55, N.4. – P. 399–432.Hermens, W.T. Viral vectors, tools for gene transfer in the nervous system / W.T. Hermens, J. Verhaagen // Prog. Neurobiol. – 1998. – Vol. 55, N.4. – P. 399–432.Isenmann, S. How to keep injured CNS neurons viable – strategies for neuroprotection and gene transfer to retinal ganglion cells / S. Isenmann, C. Schmeer, A. Kretz // Mol. Cell Neurosci. – 2004. – Vol. 26, N.1. – P. 1–16.Isenmann, S. How to keep injured CNS neurons viable – strategies for neuroprotection and gene transfer to retinal ganglion cells / S. Isenmann, C. Schmeer, A. Kretz // Mol. Cell Neurosci. – 2004. – Vol. 26, N.1. – P. 1–16.Martin, K.R. Gene delivery to the eye using adeno-associated viral vectors / K.R. Martin, R.L. Klein, H.A. Quigley // Methods. – 2002. – Vol. 28, N.2. – P. 267–275.Martin, K.R. Gene delivery to the eye using adeno-associated viral vectors / K.R. Martin, R.L. Klein, H.A. Quigley // Methods. – 2002. – Vol. 28, N.2. – P. 267–275.Surace, E.M. Adeno-associated viral vectors for retinal gene transfer / E.M. Surace, A. Auricchio // Prog. Retin. Eye Res. – 2003. – Vol. 22, N.6. – P. 705–719.Surace, E.M. Adeno-associated viral vectors for retinal gene transfer / E.M. Surace, A. Auricchio // Prog. Retin. Eye Res. – 2003. – Vol. 22, N.6. – P. 705–719.Aramant, R.B. Progress in retinal sheet transplantation / R.B. Aramant, M.J. Seiler // Prog. Retin. Eye Res. – 2005. – Vol. 23, N.5. – P. 474–494.Aramant, R.B. Progress in retinal sheet transplantation / R.B. Aramant, M.J. Seiler // Prog. Retin. Eye Res. – 2005. – Vol. 23, N.5. – P. 474–494.Retinal incorporation and differentiation of neural precursors derived from human embryonic stem cells / E. Banin, A. Obolensky, M. Idelson [et al.] // Stem Cells. – 2006. – Vol. 24, N.2. – P. 246–257.Retinal incorporation and differentiation of neural precursors derived from human embryonic stem cells / E. Banin, A. Obolensky, M. Idelson [et al.] // Stem Cells. – 2006. – Vol. 24, N.2. – P. 246–257.Crystallin--b2-overexpressing NPCs support the survival of injured retinal ganglion cells and photoreceptors in rats / M.R. Böhm, S. Pfrommer, C. Chiwitt [et al.] // Invest. Ophthalmol. Vis. Sci. – 2012. – Vol. 53, N.13. – P. 8265-8279.Crystallin--b2-overexpressing NPCs support the survival of injured retinal ganglion cells and photoreceptors in rats / M.R. Böhm, S. Pfrommer, C. Chiwitt [et al.] // Invest. Ophthalmol. Vis. Sci. – 2012. – Vol. 53, N.13. – P. 8265-8279.Cramer, A.O. Translating induced pluripotent stem cells from bench to bedside: application to retinal deceases / A.O. Cramer, R.E. McLaren // Curr. Gene Ther. – 2013. – Vol. 13, N.2. – Р. 139–151.Cramer, A.O. Translating induced pluripotent stem cells from bench to bedside: application to retinal deceases / A.O. Cramer, R.E. McLaren // Curr. Gene Ther. – 2013. – Vol. 13, N.2. – Р. 139–151.Das, A.M. Stem cell therapy for retinal degeneration: retinal neurons from heterologous sources / A.M. Das, X. Zhao, I. Ahmad // Semin. Ophthalmol.- 2005. – Vol. 20, N.1. – P. 3–10.Das, A.M. Stem cell therapy for retinal degeneration: retinal neurons from heterologous sources / A.M. Das, X. Zhao, I. Ahmad // Semin. Ophthalmol.- 2005. – Vol. 20, N.1. – P. 3–10.Generation of Rx+/Pax6+ neural retinal precursors from embryonic stem cells / H. Ikeda, F. Osakada, K. Watanabe [et al.] // Proc. Natl. Acad. Sci. – 2005. – Vol. 102, N.32. – P. 11331–11336.Generation of Rx+/Pax6+ neural retinal precursors from embryonic stem cells / H. Ikeda, F. Osakada, K. Watanabe [et al.] // Proc. Natl. Acad. Sci. – 2005. – Vol. 102, N.32. – P. 11331–11336.Klassen, H. Stem cells and retinal repair / H. Klassen, D.S. Sakaguchi, M.J. Young // Prog. Retin. Eye Res. – 2004. – Vol. 23, N.2. - P. 149–181.Klassen, H. Stem cells and retinal repair / H. Klassen, D.S. Sakaguchi, M.J. Young // Prog. Retin. Eye Res. – 2004. – Vol. 23, N.2. - P. 149–181.Availability of pre- and pro-regions of transgenic GDNF affects the ability to induce axonal sprout growth / N. Kust, D. Panteleev, I. Mertsalov [et al.]// Mol Neurobiol. – 2014. –[Epub ahead of print].Availability of pre- and pro-regions of transgenic GDNF affects the ability to induce axonal sprout growth / N. Kust, D. Panteleev, I. Mertsalov [et al.]// Mol Neurobiol. – 2014. –[Epub ahead of print].Cell transplantation as a treatment for retinal disease / R.D. Lund, A.S. Kwan,D.J. Keegan [et al.] // Prog. Retin. Eye Res. – 2001. – Vol. 20, N.4. – P. 415–449.Cell transplantation as a treatment for retinal disease / R.D. Lund, A.S. Kwan,D.J. Keegan [et al.] // Prog. Retin. Eye Res. – 2001. – Vol. 20, N.4. – P. 415–449.Retinal transplantation: progress and problems in clinical application / R.D. Lund, S.J. Ono, D.J. Keegan, J.M. Lawrence // J. Leukoc. Biol. – 2003. – Vol. 74, N.2. – P. 151–160.Retinal transplantation: progress and problems in clinical application / R.D. Lund, S.J. Ono, D.J. Keegan, J.M. Lawrence // J. Leukoc. Biol. – 2003. – Vol. 74, N.2. – P. 151–160.Control of small inhibitory RNA levels and RNA interference by doxycycline induced activation of a minimal RNA polymerase III promoter / Amar L., Desclaux M., Faucon-Biguet N. [et al.] // Nucleic Acids Res.-2006. – Vol. 34, N.5. – P. 37.Control of small inhibitory RNA levels and RNA interference by doxycycline induced activation of a minimal RNA polymerase III promoter / Amar L., Desclaux M., Faucon-Biguet N. [et al.] // Nucleic Acids Res.-2006. – Vol. 34, N.5. – P. 37.The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site / T. Kanno, H. Yamamoto, T. Yaguchi [et al.] // J. Lipid Res. – 2006. – Vol.47, N.6. – P. 1146–1156.The linoleic acid derivative DCP-LA selectively activates PKC-epsilon, possibly binding to the phosphatidylserine binding site / T. Kanno, H. Yamamoto, T. Yaguchi [et al.] // J. Lipid Res. – 2006. – Vol.47, N.6. – P. 1146–1156.In vivo trafficking and targeted delivery of magnetically labeled stem cells / A.S. Arbab, E.K. Jordan, L.B. Wilson [et al.] // Hum Gene Ther. – 2004. – Vol. 15, N.4. – P. 351–360.In vivo trafficking and targeted delivery of magnetically labeled stem cells / A.S. Arbab, E.K. Jordan, L.B. Wilson [et al.] // Hum Gene Ther. – 2004. – Vol. 15, N.4. – P. 351–360.Using a neodymium magnet to targe delivery of ferumoxide-labeled human neural stem cells in a rat model of focal cerebral ischemia / M. Song, Y.J. Kim, Y.H. Kim [et al.] // Hum. Gene Ther. – 2010. – Vol. 21, N.5. – P. 603-610.Using a neodymium magnet to targe delivery of ferumoxide-labeled human neural stem cells in a rat model of focal cerebral ischemia / M. Song, Y.J. Kim, Y.H. Kim [et al.] // Hum. Gene Ther. – 2010. – Vol. 21, N.5. – P. 603-610.Magnetic tagging increases delivery of circulating progenitors in vascular injury / P.G. Kyrtatos, P. Lehtolainen, M. Junemann-Ramirez [et al.]// JACC Cardiovasc. Interv. – 2009. – Vol. 2, N. 8. – P. 794-802.Magnetic tagging increases delivery of circulating progenitors in vascular injury / P.G. Kyrtatos, P. Lehtolainen, M. Junemann-Ramirez [et al.]// JACC Cardiovasc. Interv. – 2009. – Vol. 2, N. 8. – P. 794-802.Thickness sensing of hMSCs on collagen gel directs stem cell fate / W.S. Leong, C.Y. Tay, H. Yu [et al.] // Biochem. Biophys. Res. Commun. – 2010. – Vol. 401, N.2. – P. 287–292.Thickness sensing of hMSCs on collagen gel directs stem cell fate / W.S. Leong, C.Y. Tay, H. Yu [et al.] // Biochem. Biophys. Res. Commun. – 2010. – Vol. 401, N.2. – P. 287–292.Focused magnetic stem cell targeting to the retina using superparamagnetic iron oxide nanoparticles / A. Yanai, U.O. Häfeli, A.L. Metcalfe [et al.] // Cell Transplant. – 2012. – Vol. 21, N.6. – Р. 1137–1148.Focused magnetic stem cell targeting to the retina using superparamagnetic iron oxide nanoparticles / A. Yanai, U.O. Häfeli, A.L. Metcalfe [et al.] // Cell Transplant. – 2012. – Vol. 21, N.6. – Р. 1137–1148.Intraocular gene transfer of ciliary neurotrophic factor prevents death and increases responsiveness of rod photoreceptors in the retinal degeneration slow mouse / M. Cayouette, D. Behn, M. Sendtner [et al.] // J. Neurosci. – 1998. – Vol. 18, N.22. – P. 9282–9293.Intraocular gene transfer of ciliary neurotrophic factor prevents death and increases responsiveness of rod photoreceptors in the retinal degeneration slow mouse / M. Cayouette, D. Behn, M. Sendtner [et al.] // J. Neurosci. – 1998. – Vol. 18, N.22. – P. 9282–9293.Hoffman, L.M. Cell-mediated immune response and stability of intraocular transgene expression after adenovirusmediated delivery / L.M. Hoffman, A.M. Maguire, J. Bennett // Invest. Ophthalmol. Vis. Sci. – 1997. – Vol. 38, N.11. – P. 2224–2233.Hoffman, L.M. Cell-mediated immune response and stability of intraocular transgene expression after adenovirusmediated delivery / L.M. Hoffman, A.M. Maguire, J. Bennett // Invest. Ophthalmol. Vis. Sci. – 1997. – Vol. 38, N.11. – P. 2224–2233.Immune responses limit adenovirally mediated gene expression in the adult mouse eye / M.B. Reichel, R.R. Ali, A.J. Thrasher [et al.] // Gene Ther. – 1998. – Vol. 5, N.8. – P. 1038–1046.Immune responses limit adenovirally mediated gene expression in the adult mouse eye / M.B. Reichel, R.R. Ali, A.J. Thrasher [et al.] // Gene Ther. – 1998. – Vol. 5, N.8. – P. 1038–1046.

The authors declare that there are no conflicts of interest present.